For a number of reasons including ease of cell culture, genetic manipulation and experimental design the social amoeba Dictyostelium discoideum has long been a model system for investigating the physiological roles of the actomyosin cytoskeleton. One of our interests in recent years has been the functions of the actin cross-linking proteins cortexillins (ctx) I, II and III. We had shown that the double knockout of ctxI and ctxII completely inhibits, cell streaming and development by decreasing the secretion of cAMP. More recently, we have focused on ctxIII. We find that expressed ctxIII forms heterodimers with both ctxI and ctxII and the three cortexillins form one or more complexes with the Dictyostelium GAP protein DGAP1 (also called IQGAP1)but, unlike ctxI and ctxII, ctxIII does not bind to GAPA, another GAP protein. Binding of ctxIII to DGAP1 requires the presence of either ctxI or ctxII (but not both) and, thus, it seems likely that ctxIII binds to DGAP1 only as a heterodimer with either ctxI or ctxII. The C-terminal half of ctxIII, but not the N-terminal half, binds to the ctx-DGAP1 complexes, most likely as a dimer with ctxI and/or ctxII, which is reasonable as the sequence of the C-terminal half of ctxIII predicts formation of a coiled-coil helix, but with lower probability than the C-terminal halves of ctxI and ctxII. We find that, unlike ctxI and ctxII which form homodimers and crosslink actin filaments in vitro and in vivo, recombinant ctxIII is monomeric and fragments actin filaments in vitro. CtxIII-null cells are flatter than wild-type cells and actin filaments are less homogenously distributed. Expression of ctxIII in the null cells recovers the morpholgy of wild-type cells. Expressed ctxIII co-localizes with ctxI and ctxII in the cortex of vegetative amoebae and the leading edge of motile cells, but localization of ctxIII to the cleavage furrow of dividing ells requires the presence of DGPA1. Functionally, ctxIII is a negative regulator of cytokinesis, pinocytosis and phagocytosis which are enhanced in ctxIII-null cells and unhibited in cells over-expressing ctxIII. CtxIII-nul cells chemotax normally, so ctxIII is not required for cell motility, but the ctxIII-null cells form smaller mounds and smaller, but otherwise apparently normal, fruiting bodies.